Isolation of a human retrovirus

ABSTRACT

The present invention comprises compositions and methods comprising a spumavirus isolated from a human. More specifically, the spumavirus of the present invention was isolated from a human who had exposure to nonhuman primates. Importantly, the methods and compositions of the present invention comprising the spumavirus and including antibodies to the spumavirus, can be used to detect the presence of spumavirus or antibodies in body fluids, for pathogenicity studies of related viruses, and as a vector for gene therapies. The present invention can also be used for treatment of conditions in humans due to the presence of rapidly dividing cells and for recombinant live virus vaccination.

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, international application PCT/US00/16433,filed Jun. 14, 2000 (published under PCT Article 21(2) in English),which claims priority to U.S. provisional patent application Ser. No.60/139,219, filed Jun. 14,1999, which applications are incorporatedherein in their entirety by reference.

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government.

TECHNICAL FIELD

The present invention relates to a novel retrovirus, a spumavirus, thathas been isolated from a human. More particularly, the novel spumavirusmay be used as a vector for gene therapy or as a recombinant virusvaccine. The invention can also serve as a reagent in pathogenicitystudies of related viruses and be used to screen for spumavirusinfection in humans.

BACKGROUND OF THE INVENTION

Spumavirus, also known as foamy virus for the characteristics ofvacuolization the virus induces in cell culture, belongs to a distinctgroup of retroviruses. The simian foamy viruses (SFVs) include isolatesfrom Old World and New World monkeys and are classified into 10different serotypes based on serological cross-reactivities. Virusappears to persist in the host for a long period of time in a latentform and can exist in the presence of neutralizing antibody.

Currently the most studied retrovirus, human immunodeficiency virus(HIV), is believed to be derived from non-human primate transmissioninto humans at some past time. Concerns about the risk of transmissionof retroviruses from non-human primates (NHP) to humans working inresearch laboratories were heightened in the early 1990's when twopersons developed antibodies to SIV (simian immunodeficiency virus)following work-related exposures, one of whom had clear evidence ofpersistent viral infection. (See CDC Anonymous survey for simianimmunodeficiency virus (SIV) seropositivity in SIV laboratoryresearchers—United States, 1992. MMWR Morb. Mort. Wkly. Rep. 1992; 41:814-5; Khabbaz R. F., et al. Brief report: infection of a laboratoryworker with simian immunodeficiency virus. New Eng. J. Med. 1994; 330:172-7; Khabbaz R. F., et al. Simian immunodeficiency virus needlestickaccident in a laboratory worker. Lancet 1992; 340: 271-3; and CDC.Guideline to prevent simian immunodeficiency virus infection inlaboratory workers and animal handlers. MMWR 1988; 37:693-704.) Inaddition to SIV, non-human primate species used in biomedical researchare commonly infected with SFV (simian foamy virus), STLV (simian t-celllymphotrophic virus), and/or type D retroviruses. All of theseretroviruses cause lifelong infections in NHP, and some are known to betransmissible through sexual contact, blood, or breast feeding. NaturalSFV infections in non-human primates have not been definitivelyassociated with disease. In NHP, infection with the other retrovirusesmay result in a clinical spectrum ranging from asymptomatic infection tolife threatening immunodeficiency syndromes or lymphoproliferativedisorders. The transmission routes of SFVs among non-human primatesremain undefined, but the prevalence of seroreactivity is high amongcaptive adult non-human primates.

Studies of the prevalence of spumavirus infection of humans are limitedand the findings are not definitive. Though there is some evidence ofhuman infection with SFV (antibodies and positive PCR results), suchoccurrence has been reported in only two persons, both of whom hadoccupational risks for infection. Associated disease was not reported ineither. (See Schweizer M., et al. Absence of foamy virus DNA in Graves'disease. AIDS Res. & Human Retrov. 1994; 10: 601-5; Neumann-Haefelin D.,et al., Foamy viruses. Intervirology 1993; 35: 196-207; and SchweizerM., et al., Markers of foamy virus infections in monkeys, apes, andaccidentally infected humans: appropriate testing fails to confirmsuspected foamy virus prevalence in humans. AIDS Res. & Human Retrov.1995; 11: 161-70).

Other inconclusive evidence was seen in early studies which described arelatively high rate of seroreactivity to antibodies to spumavirusesamong human populations not known to be exposed to non-human primates.In some instances seroreactivity was suggestively linked to humandisease, including disorders of the central nervous system, thyroiddisease, and Chronic Fatigue Syndrome. In most instances these studieslacked definitive evidence of human infection and were not subsequentlyconfirmed (See Heneine, W., et al., Absence of evidence for humanspumaretrovirus sequences in patients with Graves' disease [letter]. J.Acq. Immune Defic. Synd. & Human Retrov. 1995; 9: 99-101; Simonsen, L.,et al.,. Absence of evidence for infection with the humanspumaretrovirus in an outbreak of Meniere-like vertiginous illness inWyoming, USA [letter]. Acta Oto-Laryngologica 1994; 114: 223-4; andHeneine, W., et al., Lack of evidence for infection with known human andanimal retroviruses in patients with chronic fatigue syndrome. Clin.Infect. Dis. 1994; 18: S121-5).

Recent publications indicate that earlier serological tests showinghuman spumavirus antibodies in the human population were incorrect.Immunological investigation of a previously reported human spumavirusrevealed that it shared common antigens in complement fixation,immunofluorescence and neutralization assays with the chimpanzee foamyvirus, SFV-6. The virus known as HFV, Human Foamy Virus was derived froma nasocarcinoma and is now believed not to be a human foamy virus, but achimpanzee virus. Failure to detect serological evidence of HFVinfection in people from a wide geographical area suggested that thisvirus isolate was a variant of SFV-6, particularly since sera fromchimpanzees naturally infected with SFV-6 neutralized both viruses. In asurvey for prevalence of HFV in more than 5000 human sera, collectedfrom geographically diverse populations, none of the serum samples wereconfirmed as positive. Taken together with sequence analysis endorsingthe phylogenetic closeness of the purported human spumavirus to SFV-6,these data strongly suggest that HIV is not naturally found in the humanpopulation. (See AH, M. et al., “No evidence of antibody to Human FoamyVirus in widespread human populations,” AIDS Research and HumanRetroviruses, Vol. 12, No. 15, 1996).

Novel human spumaviruses have been found in humans who were exposed tononhuman primates. These novel viruses are unique viruses that reproducein humans and yet cause no disease. These viruses are disclosed in U.S.Pat. No. 5,882,912 and U.S. patent application Ser. No. 60/105,811,incorporated in their entirety herein. The existence of new humanretroviruses in humans that were derived originally from simian sourcesindicates a need for compositions and methods for the immunologicalscreening of the human population for the prevalence of spumavirusinfection as web as for the screening of the human blood supply.

Recent concern that xenotransplantation, the use of living tissues fromnonhuman species in humans for medical purposes, may introduce newinfections into the human community has increased the importance ofdefining the ability of simian retroviruses to infect and/or causedisease in humans (See Chapman, L. E., et al. Xenotransplantation andxenogeneic infections. New Engl. J. Med. 1995; 333: 1498-1501; DHHS.Docket No. 96M0311. Draft Public Health Service (PHS) Guideline onInfectious

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a DNA sequence comparison between SFVHu-6 and various simianviruses isolated from chimpanzee subspecies. The numbers indicatepercent identity between SFVHu-6 and the viruses isolated fromindividual chimpanzees, source chimp B1, and other chimps the exposedperson worked with, A055, A101, A136 and A182 within the integrase, gagand ORF2 gene regions.

FIG. 2 (a and b) are phylogenetic trees showing the relationshipsbetween the integrase gene sequence of the novel spumavirus of thepresent invention and known spumaviruses from other non-human primatesand various chimpanzee subspecies, including the source chimp, B1.

FIG. 3 shows an electron micrograph of the spumavirus isolated from Case6, SFVHu-6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods and compositions comprisinga novel spumavirus, SFVHu-6. The novel spumavirus of the presentinvention has multiple utilities, in part, based on its characteristicsof an inability to cause disease in the infected human and its inabilityto transfer between an infected human and close contacts of the human.Some of these utilities include use of compositions derived from thehuman spumavirus as a reagent for the immunological screening forspumaviruses in humans, especially those who work with nonhuman primates(NHP), as well as spumavirus infection in general. The novel spumavirusof the present invention can also serve as a vector in gene therapybecause the virus appears to cause no disease in humans and is nottransmitted to other humans. The novel spumavirus is a useful candidateas a gene therapy vector. Additionally, the novel spumavirus of thepresent invention can be used as a reagent in pathogenicity studies ofthese and other related viruses. Moreover, the sequences of SFVHu-6 ofthe present invention can be used as probes to detect virus orantibodies to the virus in biological samples. Vectors include, but arenot limited to, procaryotic, eucaryotic and viral vectors. Thespumavirus of the present invention can also be used as a liverecombinant virus vaccine. Additionally, the spumavirus of the presentinvention can be used as a replicating viral system to kill livedividing cells, either in vitro or in vivo.

The spumaviruses or foamy viruses are by far the least wellcharacterized of the retroviruses. They have been isolated as agentsthat cause vacuolation (“foaming”) of cells in culture from a number ofin vitro. The present invention comprises methods and compositionscomprising recombinant live virus vaccines using SFVHu-6 as the viralvector. The present invention also comprises methods and compositionsfor detecting spumavirus or antibodies to spumavirus in biologicalfluids, tissues or organs.

Accordingly, it is an object of the present invention to providecompositions comprising a novel spumavirus.

It is another object of the present invention to provide methods ofdetecting a spumavirus.

It is yet another object of the present invention to provide methods andcompositions for detecting the presence and amount of spumavirus in abody fluid or organ.

A further object of the present invention is to provide compositions andmethods for treating genetic and physiologic disorders using genetherapy techniques comprising the novel spumavirus of the presentinvention as a vector for nucleic acid sequences and antisensesequences.

Another object of the present invention is to provide compositions andmethods useful for manipulating the expression of genes.

Yet another object of the invention is to provide vaccines.

Still a further object of the present invention is to providecompositions and methods for treating viral infections in humans oranimals.

Another object of the present invention is to provide compositions andmethods that are effective in treating genetic diseases.

An object of the present invention is to provide methods of treatingmicrobial infections in humans or animals.

It is another object of the present invention to provide for treatmentsof conditions that are caused in part by rapidly dividing cellulargrowth.

Another object of the present invention is to provide live recombinantvirus vaccines.

An object of the present invention is to provide diagnostic tools suchas antibodies or antigens for the monitoring of the blood supply ororgan and tissue donation for the presence of spumavirus.

An object of the present invention is to provide reagents forpathogenecity studies.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments and the appended claims.

Disease Issues in Xenotransplantation. Federal Register Vol. 61, No.185. Sep. 23, 1996). Currently, the primary animal species considered asdonors for xenografts are baboons and pigs, though other species may beconsidered in the future. Thus, what is needed are compositions andmethods for detecting viruses that may be transmitted from the nonhumanorgan donors to the recipient human. Additionally, information regardingthese transmissible agents may provide valuable information about theorgan donors' cellular receptors that may be important fortransplantation success.

Gene therapies have long looked for a good vector that can transport theforeign gene of choice into human cells. The lack of any known diseaseassociated with the virus of the present invention makes the presentinvention an ideal candidate for gene therapy regimens. Thus,compositions and methods for gene therapy are needed that use a vectorcapable of carrying a significant amount of foreign DNA that will enterthe host organism and not cause disease.

Compositions and methods for vaccination using recombinant liveretroviruses are also needed. A live virus, that causes no illness inhumans, and that has genes of antigens of choice incorporated into itsgenome, would provide for an excellent vaccination tool. The retroviruswould reproduce in the human host and expose the immune system toantigens so that an immune response can be initiated.

Targeted attack on reproducing cells is a goal of cancer treatment. Whatis needed are compositions and methods for cancer treatment that arespecific for dividing cells that do not cause systemic damage to thecancer patient. A virus that could infect and kill dividing cells,without killing other cells of the host would provide a solution forcancer treatment.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions comprisinga novel spumavirus or foamy virus, taxonomically named SFVHu-6. Thevirus was deposited with the American Type Culture Collection (ATCC) onDec. 2, 1998. The present invention comprises an isolated humanspumavirus that has been definitively derived from a human with nodisease. The novel spumavirus of the present invention has beenmaintained through tissue culture cells where it causes the vacuolationof the cells that is characteristic of foamy viruses.

The present invention further comprises methods and compositions for theuse of a replicating viral system to kill live dividing cells in a hostor mammalian species, including monkeys, cattle, cats, and reportedly inhumans. Persistent infection with these viruses is not associated withany known disease.

Recent studies using improved diagnostic assays have shown no evidenceof human foamy virus infection of humans in studies of large populations(approximately 8,000 persons). Given these results, the identificationof seroreactivity in six persons occupationally exposed to non-humanprimates is notable and several different novel spumaviruses have beenisolated from such workers.

Two of these human spumaviruses, SFVHu-1 and SFVHu-3, disclosed in U.S.Pat. No. 5,882,912 and PCT/US99/25171, incorporated herein in theirentirety, were isolated from two occupationally exposed workers. SFVHu-1has structural and functional similarities to a simian spumavirus ofAfrican green monkey origin while SFVHu-3 has similarities with ababoon-like simian spumavirus.

The present invention comprises the isolation and characterization of aspumavirus, SFVHu-6, that was shown to have been transmitted from anon-human primate to a human at some point in the past. The retrovirusof the present invention, unlike another retrovirus of a more virulentnature, HIV-1 (human immunodeficiency virus-type 1), is not readilytransmitted from human to human. The spumavirus of the present inventioncan be used in diagnosing spumavirus infections and used as a vector ingene therapy procedures.

The present invention also includes methods and compositions fordetecting spumavirus in biological fluids. The methods and compositions,including kits, can be in any configuration well known to those ofordinary skill in the art. The present invention also includesantibodies specific for the spumavirus and antibodies that inhibit thebinding of antibodies specific for the spumavirus. These antibodies canbe polyclonal antibodies or monoclonal antibodies, or fragments thereof.The antibodies specific for the spumavirus can be used in diagnostickits to detect the presence and quantity of spumavirus in biologicalfluids or in organs from non-human primates for xenotransplantation.Antibodies specific for spumavirus may also be administered to a humanor animal to passively immunize the human or animal against spumavirus,thereby reducing infection after accidental exposure to non-humanprimate bodily fluids.

The present invention also includes compositions and methods, includingkits, for detecting the presence and quantity of antibodies that bindspumavirus in body fluids. The methods, including kits, can be in anyconfiguration well known to those of ordinary skill in the art Such kitsfor detection of spumavirus itself or detection of antibodies to thespumavirus can be used to monitor the blood supply for the presence ofspumavirus in the blood supply.

The present invention also includes methods and compositions comprisingrecombinant live virus vaccines. Exogenous genes can be inserted intothe genome of the virus of the present invention. The genes can code forany protein or proteins for which vaccination or gene therapy isdesired. SFVHu-6 can provide a high level of antigen to the hostcarrying the virus. As an example of such use, SFVHu-6 carryingexogenous genes is administered to a human, the virus would infect thecells and replicate. The exogenous genes would be translated and wouldprovide the selected antigens to the immune system of the human. A novelaspect of such recombinant live viruses is that SFVHu-6 does not causedisease in the human. Additionally, there is no transmission from onehuman to another non-infected human, even by close contact with exchangeof bodily fluids. The recombinant live virus vaccines of the presentinvention provide one or several antigens in a most optimum method tothe immune system of the selected human.

The present invention further includes methods and compositions for theuse of a replicating viral system to kill live dividing cells in a hostor in vitro. In in vitro uses, SFVHu-6 can be used to detect and killrapidly dividing cells. Foamy viruses, including SFVHu-6, can infect awide variety of species of cells and can be used in many in vitro cellsystems. For example, if the assay of the in vitro cell system requiredthe identification of quiescent cells, application of SFVHu-6 to thetissue culture system would result in the selection of the rapidlydividing cells by SFVHu-6. All of the tissue culture cells would beinfected but only the dividing cells would be destroyed because SFVHu-6has a productive infection and its cytopathic destruction effects onlydividing cells. The remaining non-dividing cells of the culture wouldremain alive.

In a host, the ability of SFVHu-6 to infect dividing cells provides anexcellent tent for conditions due to the presence of rapidly dividingcells. For example, a person with disease due to rapidly dividing cells,such as cancer or any known angiogenic condition such asangiogenesis-dependent diseases, could be infected with SFVHu-6. Suchvirus may or may not carry other, exogenous genes for other effects inthe host. Because SFVHu-6 does not cause disease in humans and there isno transmission of the virus to close contacts with humans, only theperson with the disease due to rapidly dividing cells will be treated.The virus will infect the rapidly dividing cells and kill them. Forexample, a person with a fast growing tumor would be infected withSFVHu-6 and the cells of the tumor would be destroyed by the virus. TheSFVHu-6 can be recombinantly modified, for example, to be selective forcellular receptors on the tumor to make the virus even more specificallytargeted to just those cells.

Such treatment with SFVHu-6 could be used for any condition in whichrapidly dividing cells provide an aspect of the pathology of thecondition. One such condition is the presence of uncontrolledangiogenesis within the body. Angiogenesis-dependent diseases are wellknown in the art and are caused in part by the rapid growth of bloodvessels.

In response to the identification of simian immunodeficiency virus (SIV)infection in persons who work with simians, or other occupationallyexposed workers, Centers for Disease Control and National Institutes forHealth collaborated in an anonymous serosurvey of persons with similarwork exposures. Simian immunodeficiency virus seroreactivity was presentin 3/427 (0.7%) stored serum samples from these anonymous workers (SeeCDC, Anonymous survey for simian immunodeficiency virus (SIV)seropositivity in SIV laboratory researchers—United States, 1992. MMWR,Morb. Mort. Wkly. Rep. 1992; 41: 814-5; Khabbaz R. F., et al.,. Briefreport: infection of a laboratory worker with simian immunodeficiencyvirus. New Eng. J. Med. 1994; 330: 172-7). Consequently, a voluntarytesting and counseling program was developed that allowed linkagebetween specific exposures or health outcomes and serostatus of personsoccupationally exposed to simian immunodeficiency virus. The workersenrolled in this voluntary, limp prospective simian immunodeficiencyvirus surveillance are also at occupational risk for exposure to otherretroviruses common in NHP.

In 1995, the linked surveillance was expanded to include voluntarytesting and counseling for exposure to simian spumaviruses (morecommonly called simian foamy viruses, or SFV), simian T-lymphotropicviruses (STLV), and simian type D retroviruses (STRV). 1,823 samplesfrom 13 institutions in the United States had been tested for simianimmunodeficiency virus; samples from 231 of the participating volunteerworkers were also tested for other retroviruses from non-human primates.Four of these 231 workers (1.7%) were determined to be infected with aSFV-hike virus by serology and PCR.

A seroprevalence of 3% (4/133) was found by Western blot analysis of zooworkers exposed to mammals including NHP. None of 189 unexposed zooworkers was SFV-positive Workers occupationally exposed to NHP in zoosor prima research are at risk of SFV zoonosis, primarily fromchimpanzees, and thus represent a unique population to study thepathogenetic potential and transmissibility of zoonotic SFV infections.

An immunofluorescent assay that was developed using cells infected witha chimpanzee foamy virus, SFV-6cpz, identified antibodies to a SFV- likevirus in recently collected serum specimens from a worker (Case 6). Thespecimens were also Western blot positive, showing reactivity to bothp70 and p74 gag precursor bands of SFV-6cpz antigen. Repeat testing ofadditional sera obtained from this worker at later time points are alsopositive in both assays.

Additional blood samples from Case 6 were tested for SFV proviral DNAsequences using polymerase chain reaction (PCR) assays employing primersets from two regions of the polymerase gene that are conserved amongknown primate foamy viruses. All samples were PCR positive in bothregions. The PCR products from three regions (the gag, integrase andORF2 gene) were sequenced. There was 93-100% identity between the virussequences determined from the SFV-infected source chimp (SEQ ID 2, 4, 6)and the virus sequences determined from the human, Case 6, SFCHu-6 (SEQID 1, 3, 5). This near sequence identity confirms that the virusoriginated in chimpanzee B1 and was transmitted to Case 6. Thecorresponding RNA sequences and resulting proteins can be deduced fromthese sequences, and are included within the scope of the presentinvention.

SEQ. ID 1 comprises 613 nucleotides of the gag gene of SFVHu-6, SEQ ID 3comprises 425 nucleotides of the int (integrase) gene of SFVHu-6, andSEQ ID 5 comprises 240 nucleotides of the ORF 2 of SFVHu-6. SEQ ID 7comprises the 3' part of the env (envelope) gene, the complete ORF1 andORF2 and the 5' end of the 3' LTR of SFVHu-6. SEQ. ID 2 comprises 616nucleotides of the gag gene of the virus isolate from B1, SEQ. ID 4comprises 425 nucleotides of the integrase gene of the virus isolatefrom B1, and SEQ. ID 6 comprises 240 nucleotides of the ORF2 of thevirus isolate from B1.

Case 6 was severely bitten by chimpanzee B1 in 1977. Chimpanzee B1 iscurrently in good health. A serum sample obtained from chimpanzee B1 wasfound to be positive for SFV-6antibodies. SFV-6sequences from theperipheral blood lymphocytes (PBL) of B1 and from four otherSFV-infected chimpanzees from the same facility were amplified andcompared to Case 6. Sequences from Case 6 and chimpanzee B1 wereindistinguishable (100% identity) in both the integrase and gag regions.In contrast, the SFV integrase and gag sequences from the four controlchimpanzees were 92.7 to 93.6% and 84.4 and 84.7% homologous to those ofCase 6, respectively. See FIG. 1 for a chart of the comparison. Theobserved identity in the SFV-6sequences reflects the high geneticstability of SFV-6, a characteristic seen in human/simian Tlymphotrophic viruses rather than in HIV/SIV infections. This geneticstability makes the present invention uniquely well suited for genetherapy uses.

The discovery of subspecies-specific diversity in SIV from chimpanzees(SIVcpz) raised the possibility that a similar evolution of SFV inchimpanzee subspecies might explain the sequence difference between Case6 and the control chimpanzees. Thus, the subspecies of all livechimpanzees was determined by mitochondrial DNA analysis. B1 was foundto be Pan troglodytes troglodytes (P. t. troglodytes) while the otherfour control chimpanzees, infected with the closely related SFV,belonged to Pan troglodytes verus (P. t. verus). The relationshipbetween the SFVHu-6 isolate and other known spumaviruses is shown inFIG. 2 (a and b) which is a phylogenetic tree based on the homology ofthe nucleotide sequences of these viruses. Phylogenetic analysis usingthe integrase sequences clearly indicates that the sequence pair fromCase 6 and chimpanzee B1 fell within the clade of chimpanzee sequencesbut did not cluster with any other sequences including those from thefour control chimpanzees. Thus, the source of the SFV in Case 6 ischimpanzee B1. The identification of this primary zoonotic SFV infectionprovides a unique opportunity to study the early events of retrovirusadaptation to the human host.

Case 6 provides a rare opportunity to examine SFV genome stabilityduring both zoonotic transmission and persistent human infection. SFVendemic to different species of non-human primates demonstrates thegreatest level of genome sequence diversity within the U3 region of thelong terminal repeat (LTR) and the 3' accessory open-reading frames(ORF), suggesting that adaptive changes may occur during zoonosis. Infoamy viruses, the LTR aids in the replication of the virus. The LTR istransactivated by a virus-specific protein, and unlike a relatedretrovirus, HIV, no human cellular transcription factors activate thevirus. LTRs in retroviruses like HIV have conserved consensus sequencesfor cellular transcription factors.

It is known that another human-derived spumavirus has stable, conservedLTRs and internal promoters, providing conserved transcriptionallyimportant regions in such viruses. The sequence analysis shown in FIG. 1indicates that there is little genetic variation that occurs duringcross species transmission of SFV to humans. Thus, for gene therapyuses, this stability indicates that the virus is not acquiring humansequences that would cause it to possibly become virulent or at leastcause disease in humans due to introduced mutations. With such conservedregions, SFVHu-6, is an excellent vector, vaccine or gene therapy agentfor humans. This stability is surprising in light of the highinstability of the LTR of the virus known as HFV(Human Foamy Virus). HFVwas derived from a nasocarcinoma and is now believed not to be a humanfoamy virus, but a chimpanzee virus. The HFV LTR is unstable and hasmany deletions, thus making it an undesirable vector.

To date, zoonotic transmission of NHP retroviruses to humans has onlybeen substantiated by indirect evidence such as phylogentic relatednessbetween NHP and human retroviruses. The present invention is the firsten direct evidence for a chimpanzee to-man retroviral zoonosis. Todetermine the species origin of SFVHu-6, two PBL-derived FV sequenceswere compared with prototype SFV sequences from different non-humanprimate species. These sequences represent conserved (integrase) orvariable (gag) genomic regions among SFV . The results indicated thatboth sequences had the highest homology to SFV from chimpanzees(approximately 93% and 85% for the integrase and gag sequencesrespectively). Thus the foamy virus infection in Case 6 is of chimpanzeeorigin.

SFVHu-6 is found in the PBL of the host and is cultured from such cellsin tissue culture systems. Reverse transcriptase activity has been foundin the PBL and plasma of the infected host. Virus isolation of SFVHu-6was accomplished by co-culturing the PBL of the person identified asCase 6 with Canine thymocyte (Cf2th) cells. Importantly, this isolationhas identified a cell line that is a susceptible host cell line forisolating SFVHu-6 and other chimpanzee-like spumaviruses. Reversetranscriptase activity was detected in co-cultures from the cellsexposed to Case 6 PBLs but not from controls. Transfer of supernatantfrom the above cells exposed to Case 6's PBL passed this reversetranscriptase activity to uninfected cells, which subsequently showedcytopathic effect (CPE). This finding indicated that the infectiousagent in Case 6's PBL was transmitted to tissue culture cells which wereused to transfer the infectious agent into other tissue culture cells.Additionally, this indicated that the infectious agent reproduced in theCanine thymocyte (Cf2th) cells. DNA-PCR of infected cells was found tobe positive for a SFV-like virus. Infected cells showed strongreactivity with Case 6's sera by both immunofluorescent assay andWestern blot and no reactivity with normal sera controls. By electronmicroscopy, infected Canine thymocyte (Cf2th) cells, derived from cellfree supernatants from cells infected by exposure to infected PBL,showed a morphology characteristic of foamy virus infection (See FIG.3).

Th present invention is directed to compositions and methods comprisinga new spumavirus, SFVHu-6. The virus was isolated from a human who had asevere injury from interaction with a chimpanzee, along with exposure tomany other non-human primates. The new spumavirus, or foamy virus, doesnot appear to cause any disease in human hosts. The new virus of thepresent invention is an excellent vector for gene therapy and forvaccination purposes. Additionally, the antibodies or other detectionmethods for detecting the new virus can be used to detect the presenceof this and related viruses. In addition, the novel spumavirus of thepresent invention can be used as a reagent in pathogenicity studies ofthese and related viruses. Moreover, the sequences of the novelspumavirus of the present invention can be used as probes to detectvirus in biological samples. Vectors include but are not limited toprocaryotic, eucaryotic and viral vectors.

The sequences of SEQ ID 1-6 can be used for all the molecular biologicaltechniques known to those skilled in the art. Such uses include, but arenot limited to, generation of probes and vectors containing thesequences, antisense sequences derived from such sequences, RNAsequences such as antisense RNA, ribozyme RNA, decoy RNA, and proteinssynthesized using the sequences. RNA and other nucleic acid derivativesare contemplated by the present invention. Spumaviruses can toleratelarge deletions and still remain infectious. Such deletion sites can beused as the sites of insertion of exogenous sequences that arecontemplated by the present invention. Additionally, exogenous sequencesmay be inserted without deletions.

Many new and potentially useful technologies are being developed whichuse viral vectors and may form the basis of future medical cures andtherapies. Examples of such technologies include, but are not limitedto, gene replacement, antisense gene therapy, in situ drug delivery,treatment of cancer or infectious agents, and vaccine therapy. However,to be successful, these technologies require an effective means for thedelivery of the genetic information across cellular membranes. SFVHu-6can function as a vector to carry such genes when infecting cells.

The recent advent of technology, and advances in the understanding ofthe structure and function of many genes makes it possible toselectively turn off or modify the activity of a given gene. Alterationof gene activity can be accomplished many ways. For example,oligonucleotides that are complementary to certain gene messages or vialsequences, known as “antisense” compounds, have been shown to have aninhibitory effect against viruses. By creating an antisense compoundthat hybridizes with the targeted RNA message of cells or viruses thetranslation of the message into protein can be interrupted or prevented.In this fashion, gene activity can be modulated.

The ability to deactivate specific genes provides great therapeuticbenefits. For example, it is theoretically possible to fight viraldiseases with antisense molecules that seek out and destroy viral geneproducts. In tissue culture, antisense oligonucleotides have inhibitedinfections by herpes-viruses, influenza virus and the humanimmunodeficiency virus that causes AIDS. It may also be possible totarget antisense oligonucleotides against mutated oncogenes. Antisensetechnology also holds the potential for regulating growth anddevelopment. However, in order for the gene therapy to work, antisensesequences must be delivered across cellular plasma membranes to thecytosol.

Gene activity is also modified using sense DNA in a technique known asgene therapy. Defective genes are replaced or supplemented by theadministration of “good” or normal genes that are not subject to thedefect. Instead of being defective, the genes have been deleted, thusreplacement therapy would provide a copy of the gene for use by thecell. The administered normal genes can either insert into a chromosomeor may be present as extracellular DNA and can be used to produce normalRNA, leading to production of the normal gene product. In this fashiongene defects and deficiencies in the production of a gene product may becorrected.

Still further gene therapy has the potential to augment the normalgenetic complement of a cell. For example, it has been proposed that oneway to combat HIV is to introduce into an infected person's T cells agene that makes the cells resistant to HIV infection. This form of genetherapy is sometimes called “intracellular immunization.” Geneticmaterial such as a polynucleotide sequence may be administered to amammal in a viral vector to elicit an immune response against the geneproduct of the administered nucleic acid sequence. Such gene vaccineselicit an immune response in the following manner. First, the vialvector containing the nucleic acid sequence is administered to a humanor animal. Next, the administered sequence is expressed to form a geneproduct within the human or animal. The gene product inside the human oranimal is recognized as foreign material and the immune system of thehuman or animal mounts an immunological response against the geneproduct. The virus of the present invention may be used as a viralvector to provide the foreign nucleic acid sequences to theintracellular metabolic processes.

Additionally, gene therapy may be used as a method of delivering drugsin vivo. For example, if genes that code for therapeutic compounds canbe delivered to endothelial cells, the gene products would havefacilitated access to the blood stream. Additionally, cells could beinfected with a retroviral vector such as the present invention carryingnucleic acid sequences coding for pharmaceutical agents that preventinfection from occurring in the retrovirally infected cells.

The novel spumavirus of the present invention can also be used as a safeand effective vaccine agent. Exogenous genetic sequences for immunogenicproteins or polypeptides or antigenic fragments from a variety ofinfectious agents can be incorporated into the foamy virus RNA (thegenome of the virus). Once inside a cell, the gene product is expressedand releases the immunizing peptide to the body's immune system. Inanother method, the virus of the present invention can be used toimmunize the body against cell markers found on cancer or tumor cells.The genetic sequence of the cancer cell marker is incorporated into thefoamy virus nucleic acids and after infection with the virus, theexpressed gene product stimulates the immune system. The patient'simmune system is used to remove the cancerous cells, obviating the needfor chemotherapeutic methods.

The nucleic acid sequences of the virus of the present invention, SEQ ID1-7, can be used in a variety of methods, including, but not limited to,PCR assays. Additionally, the sequences can be used to easily detect thepresence of chimpanzee-like spumaviruses, similar to SFVHu-6, in organs,tissues or cells. The sequences can also be used to test fortransmission of spumaviruses in animals who are in contact withnon-human primates. For example, zoo workers show the greatestinactivity to antigens from chimpanzee foamy virus (FIG. 3). Thus thesezoo workers and other animal care workers in non-human primates centerscan be tested routinely for exposure to spumaviruses from variousnon-human primates. This facilitates comprehensive pathogenecity studiesin which the workers could be tested for the transmission of virusesfrom the animals cared for by the caretaker. Thus, the invention canserve as both a reagent in pathogenicity studies of related viruses andbe used to screen for spumavirus infection in humans.

The antibodies of the present invention can be used to detect thepresence of the virus or viral particles of the present invention inbody fluids or tissues. These antibodies can be raised in classicalways, such as immunination of animals with virus proteins, to generateantibodies, or through development of monoclonal antibodies usingimmunological or molecular biological techniques. These antibodies orantibody fragments can be used in diagnostic or screening kits to assessthe presence of the virus, for example, in clinical specimens.Additionally, the antibodies can be used to screen organs from non-humanprimates that may be used in humans. Detection of the presence of avirus that is transmitted from non-human primates to humans would becrucial in providing virus-free organs for transplantation.

Additionally, the ability to screen for the presence of virus orantibody to virus in animal care workers will provide methods formonitoring the viral status of such workers. If an animal care worker isexposed to the animal's bodily fluids, and viral transmission ispossible, then the antibodies of the present invention can be used topassively immunize such workers.

The virus of the present invention can be used for the treatment ofconditions due to the presence of rapidly dividing cells. In a host, theability of SFVHu-6 to productively infect dividing cells provides anexcellent treatment for conditions due to the presence of rapidlydividing cells. For example, a person with disease due to rapidlydividing cells, including but not limited to cancer or any knownangiogenic condition, could be infected with SFVHu-6. Such virus may ormay not carry other exogenous genes for other effects in the host.Because SFVHu-6 does not cause disease in the host and there is notransmission of the virus to contacts with the host, only the personwith the condition due to rapidly dividing cells win be treated. Inaddition, only the rapidly dividing cells of that host person will beproductively infected by SFVHu-6. Other cells in the body may beinfected but will not be killed because the infection in non-dividingcells is not productive. The virus will productively infect the rapidlydividing cells and kill them. For example, a person with a fast growingtumor would be infected with SFVHu-6 and the cells of the tumor would bedestroyed by the virus. Additionally, the virus may be given to a personprior to the person developing a condition caused by dividing cells, andwhen the cells begin dividing, the virus would then undergo a productiveinfection and kill the cells. This therapy may halt or inhibit suchconditions as leukemia or angiogenesis dependent diseases such asmacular degeneration.

Such treatment with SFVHu-6 could be used for any condition in whichrapidly dividing cells provide an aspect of the pathology of thecondition. One such condition is the presence of uncontrolledangiogenesis within the body. Angiogenesis dependent diseases are wellknown in the art and are caused in part by the rapid growth of bloodvessels. Another such condition is cancer or tumor growth. Cancer ortumors include both solid tumors and other types. Infection with thevirus of the present invention, which causes no disease and does noteffect the host systemically, is an improvement over currently knowntreatments that involved systemically administered agents. Suchchemotherapeutic agents kill rapidly dividing cells but also causetrauma to the entire person.

In contrasts, treatments of cancer with the present invention are not asharmful to the patient. Unlike HFV, the present invention was notderived from a patient suffering from a carcinoma and thus does not posea danger to the patient. The virus can either be administeredsystemically or injected in situ into the tumor. The virus will onlyreplicate in rapidly dividing cells and will not effect cells that arenot dividing. The infected cells are killed and tumor growth is stopped.The virus may be administered in one treatment or in a series oftreatments.

The SFVHu-6 of the present invention can be recombinantly modified to beselective for cellular receptors on the tumor to make the virus evenmore specifically targeted to just those cells. Additionally, the virusmay have altered promoter regions that can be selectively activated tocause a productive infection. The combination of different levels ofcontrol of the virus, both natural and recombinantly produced, arecontemplated in the present invention. A virus could be made specificfor attachment to only certain types of cellular receptors, for thosecells that are dividing, and will only undergo replication if anotherexogenous promoter factor is present. Viral infection by two or moreindividually defective viruses, that require factors or promoterssupplied by other foamy viruses or any type of virus, could provide formany levels of control of infection or treatment of specific conditions.

The virus may be administered to the host, for cancer treatment, genetherapy or vaccination by any methods known to those skilled in the art.Such methods include but arm not limited to injection, inhalation,ingestion, topical administration and implantation. The virus may bekilled or live, depending on the treatment considered. In vitro uses ofthe virus, sequences, vectors or probes are contemplated by the presentinvention.

The virus of the present invention does not cause disease and does notappear to be transmitted by close household contacts or sexual contacts.This belief is supported by the epidemiology data, the PCR andsequencing data, and the serology data. The isolate from Case 6,SFVHu-6, was deposited with the ATCC, under the Budapest Treaty on Dec.2, 1998, and was assigned ATCC No. VR-2635.

The present invention is further described by the examples which follow.Such examples, however, are not to be construed as limiting in any wayeither the spirit or scope of the present invention. In the examples,all parts are parts by weight unless stated otherwise.

EXAMPLES Example 1

Peripheral blood lymphocytes (PBLs) were isolated from Case 6 and werecultured with IL-2 for 48 hours, in RPMI media with 10% fetal calfserum, and pen-strep antibiotics. After 48 hours, the PBLs were added tothe Canine thymocyte (Cf2th) cells and co-cultured for 2-4 weeks. Thecells were in DMEM supplemented with 2% non-essential amino acids, 20%fetal calf serum and pen-strep antibiotics. 1 mL supernatants werecollected from the cell cultures every 3 to 4 days and tested foramp-reverse transcriptase. Procedures for PBL treatment, culturing ofCanine thymocyte (Cf2th) cells and Amp reverse transcriptase activitywere procedures known to those in the art. For example, see Heneine, W.,et al. “Detection of reverse transcriptase by a highly sensitive assayin sera from persons infected with HIV-1.” (1995). J. InfectiousDiseases, 171:1201-6.

Example 2

Because of the positive Amp-reverse transcriptase activity from cellsfrom Case 6, peripheral blood lymphocytes from Case 6 were cultured withIL-2 for 48 hours prior to addition to Canine thymocyte (Cf2th) cells.Supernatants were collected every 3 to 4 days and tested for Amp-reversetranscriptase activity. Cultures were also screened for infection ofCanine thymocyte (Cf2th) by PCR amplification and probing for SFV-likeDNA sequences. Each time the 1 mL sample of supernatant was taken forAmp reverse transcriptase activity, a 5 mL sample of supernatant wastaken and frozen at −80° C. in order to preserve a sample of the virusproducing the Amp-reverse transcriptase activity.

At day 7, Amp-reverse transcriptase testing showed a slightly positivesignal in the Canine thymocyte (Cf2th) cell culture. The Caninethymocyte (Cf2th) cells were obtained from the American Type CultureCollection (Rockville, Md.). The Amp-reverse transcriptase activityincreased over time. At the peak of Amp-reverse transcriptase activitycell-free supernatants were transferred to fresh Canine thymocyte(Cf2th) cells growing at 2×10⁵ cells/mL. At day 4 in the new culture,cytopathic effects and syncytia was observed. Transmission electronmicroscopy showed vial particles in and around the cells (See FIG. 8).Viral particles were isolated from these cultures and were stored at theCenters for Disease Control and were deposited at the ATCC.

The activity in control Cf2Th cells that were treated as above, exceptfor exposure to normal PBL instead of infected PBL, was also determined.There was no Amp-reverse transcriptase activity inherently in theseCanine thymocyte (Cf2th) cells, providing evidence that there was nocontamination by a retrovirus or spumavirus by the tissue culture cells.

Example 3

Case 6 has worked with non-human primates for more than 25 years. In1977, Case 6 incurred a severe bite from a chimpanzee (B1) that requiredsurgery and hospitalization. Retrospective analysis of twenty samples ofsera archived from Case 6 between 1984 and 1988 showed the sera to haveantibodies to SFVHu-6. Case 6 is in good health even after 14 years ofdocumented SFVHu-6 infection. A serum sample recently acquired from Case6 tested positive for SFVHu-6 antibodies by a Western blot assay. PCRanalysis of PBL DNA was positive for two SFVHu-6 sequences from the gagand intergrase viral regions. Case 6's spouse tested negative forSFV-like infection by both serologic and PCR analysis despite longexposure to the SFVHu-6infected partner. The lack of sexual transmissionor disease observed to date suggest a benign endpoint SFVHu-6 infection.

Having thus described the invention, numerous changes and modificationsthereof will be readily apparent to those having ordinary skill in theart, without departing from the spirit or scope of the invention.

7 1 613 DNA Foamy retrovirus 1 caatagatgg agtatttcct gttacaacaccagatctaag gtgcagaatt attaatgcta 60 tactaggagg aaacttaggg ttatcattaaccccagcaga ctgtgtaaca tgggactctg 120 cagtaggcac actatttgta agaacccatggacaatttcc aatgcatcag cttgggactg 180 taatacaagg aatagttaac caagaaggagtggcaacagc atatactttg ggaatgatgc 240 tttctggaca aaattatcca ttagtctcaggaattattcg gggatatttg cctggacaag 300 ctgtagtaac tgctttacaa cagcgcctagatcaagaagt agacgatcaa gcgcgagcag 360 aaacctttat tcaacatcta aatgctgtatatgaaatttt aggccttaat gccagaggac 420 agagtatacg tgcttcagtg actcctcagccccgaccgtc tagaggtaga ggtcgaggcc 480 aaagtactcc tagaccctct caaggaccagctagtagcgg acgtggacga cagcgtcctg 540 cttctggtca atacgacaga ggatctaataatcaaaatca aaatcaagga aatacaagtc 600 aaggaggata taa 613 2 616 DNA Foamyretrovirus 2 cagcaataga tggagtattt cctgttacaa caccagatct aaggtgcagaattattaatg 60 ctatactagg aggaaactta gggttatcat taaccccagc agactgtgtaacatgggact 120 ctgcagtagg cacactattt gtaagaaccc atggacaatt tccaatgcatcagcttggga 180 ctgtaataca aggaatagtt aaccaagaag gagtggcaac agcatatactttgggaatga 240 tgctttctgg acaaaattat ccattagtct caggaattat tcggggatatttgcctggac 300 aagctgtagt aactgcttta caacagcgcc tagatcaaga agtagacgatcaagcgcgag 360 cagaaacctt tattcaacat ctaaatgctg tatatgaaat tttaggccttaatgccagag 420 gacagagtat acgtgcttca gtgactcctc agccccgacc gtctagaggtagaggtcgag 480 gccaaagtac tcctagaccc tctcaaggac cagctagtag cggacgtggacgacagcgtc 540 ctgcttctgg tcaatacgac agaggatcta ataatcaaaa tcaaaatcaaggaaatacaa 600 gtcaaggagg atataa 616 3 425 DNA Foamy retrovirus 3aattattaca gggtcaaaat gtaaaaggat atcctaaaca atatacatac tttttagaag 60atggcaaagt aaaagtttcc agacctgaag gggttaaagt tattccccca caatcagacc 120gacaaaaaat agtgcttcaa gcccataatt tagcccacac cggacgtgaa gccactcttt 180taaaaattgc caacctttac tggtggccaa atatgaggaa ggatgtggtt aaacaactag 240gacgttgcca gcagtgttta atcacaaatg cttccaacaa agcctctggt cccatattaa 300gaccagatag gcctcaaaag ccttttgata aattttttat tgattatatt ggacctttgc 360caccttcaca aggatatctt tatgtattag ttgttgttga tggaatgaca ggatttacat 420ggtta 425 4 425 DNA Foamy retrovirus 4 aattattaca gggtcaaaat gtaaaaggatatcctaaaca atatacatac tttttagaag 60 atggcaaagt aaaagtttcc agacctgaaggggttaaagt tattccccca caatcagacc 120 gacaaaaaat agtgcttcaa gcccataatttagcccacac cggacgtgaa gccactcttt 180 taaaaattgc caacctttac tggtggccaaatatgaggaa ggatgtggtt aaacaactag 240 gacgttgcca gcagtgttta atcacaaatgcttccaacaa agcctctggt cccatattaa 300 gaccagatag gcctcaaaag ccttttgataaattttttat tgattatatt ggacctttgc 360 caccttcaca aggatatctt tatgtattagttgttgttga tggaatgaca ggatttacat 420 ggtta 425 5 240 DNA Foamyretrovirus 5 ttatgtttta aagtaatcta tgaaggagct atgagtcaaa aacaagaacaaaagagctgg 60 ctatgtagat taggacatgg ccatcgcatg ggggcttatg aatatcgcagaatagattta 120 tatgctatga aaaagggaaa agaaaacccc tatggagaaa ggggagatgtagctttgcaa 180 tatgcttatc aggttaaaag aggctgtaaa gcaggatgct tagcttcacaagtgcttaac 240 6 240 DNA Foamy retrovirus 6 ttatgtttta aagtaatctatgaaggagcc atgggtcaaa aacaagagca aaaaagctgg 60 ctatgcaggc taggacatggccaccgtatg ggtgcttatg actatcgcag agtagattta 120 tatgctatga aaaagggaaaagaaaacccc tatggagaaa ggggagatgt agctttgcaa 180 tatgcttatc aggttaaaagaggctgcaaa gcaggatgct tagcttcacc agtgcttaat 240 7 3576 DNA Foamyretrovirus 7 tatttacatc ttgaagactg cagaagacaa gattatgtca tatgtgatgtggtaaagata 60 gtacagcctt gtggcaatag ctcagacacg agtgactgtc ctgtctgggctgaggctgta 120 aaagaaccat ttgtgcaagt gaatcctctg aaaaacggaa gttatctggttttagcaagc 180 tccactgact gccagatccc accatatgtt cctagcattg tgactgttaacgaaacaaca 240 tcgtgttatg gactggactt taaaaggcca ctagttgcgg aagaaagattgagctttgag 300 ccacgactgc caaatctaca gctcagatta ccacatttgg taggaattattgcaaaaatt 360 aaagggataa aaatagaagt tacatcctct ggagaaagta taaaagaccagattgaaaga 420 gcaaaagctg agcttcttcg tctggacatc cacgaaggag atactcctgcctggatacaa 480 caactcgctg cagcaacaag agccgtttgg ccagcagccg cctctgctctacaaggaatt 540 gggaactttc tatctggggc tgcccaagga atatttggaa ctgcctttagtattttggga 600 tatttaaagc ctatcctcat aggtgtgggg gtcattcttt tgatcattcttatatttaag 660 attgtatcat ggattcctac caagaagaag aatcagtagc ctccacctctggcattcaga 720 acctgcagac tcttagtgag cttgttggtc ctgaaaatgc tggagagggagagctagtta 780 ttgctgaaga acctgaagaa aatcctcggc gtcctaaaag atacactaaaagagaagtta 840 aatgtgtatc ctaccatgcc tatagagaac ttgaggaaaa acatcctcaacacatcaagc 900 tccaagactg gattcccaca cctgaagaaa tgagtaagtc actttgtacaagactaatct 960 tatgtggact atatagtgca gagaaggcag gggaaatatt acggatgccttttacagtat 1020 cttgggaaca atcagacact gactctaaat gttttattgt gagttacacatgtatattct 1080 gtgatgctat aatacatgac cctatgccca taatgtggga tcctgaggtcaagatatggg 1140 taaaatataa acccctcaga ggaattgttg gatctgctgt gtttatcatgcataaacatc 1200 aaagaaactg ttcttttgtt aaaccttcta ctagttactc agaaggtccaaaaccaagac 1260 ctaggcacga tcctgtcctt cgatgtgaca tgtttgaaaa gcatcacaagcctcgggaga 1320 aacgacccag gaaacgatcc atcgataatg agtcatgtgc ttccagtagtgacaccttgg 1380 ccaatgagcc aggatcacta tgcaccaacc ctctttggaa tcctggatcactactacaag 1440 gagtgcttga agaatccagc aacttttcaa acttggaagt tcacatgtcaggtggaccct 1500 tctgggaaga ggtttatggg gactcaattt tgggtccccc ctctgggtcaggtgaacatt 1560 cagttttata aaaattacca aattttaact tgctgtcagg ctgtagatccatttgctaat 1620 atctttcatg gtactgatga tgaaatgtat gatattgatt caggacctgatgtttggtgt 1680 accccttctt tatgttttaa agtaatctat gagggagcta ttggtcaaaaacaagaacaa 1740 aagagctggc tatgtagatt aggacatggc catcgcatgg gggcttatgaatatcgcaga 1800 atagatttat atgctatgaa aaagggaaaa gaaaacccct atggagaaaggggagatgta 1860 gctttgcaat atgcttatca ggttaaaaga ggctgtaaag caggatgcttagcttcacaa 1920 gtgcttaact tcaaagctct gcagttccac agaaccctta tggctgacctcaccaatcct 1980 agattggaga gggacatctt gcctcatggc tatcaggcag ctatggaagcttatggacct 2040 cagagaggaa gtagcgagga gagggtgtgg tggaatgcca ctagaaatcaaggaagagat 2100 ggggagtatt acagagaagg aggtgaagag cctcattatc cgaatactccagcccctcat 2160 aaaaagacct gggatgaaag acataaggtc cttaagttgt cctcattcgctactccctct 2220 gacatccaac gctgggctac cagagcactg ccatatggct ggaaagtagttactgaggca 2280 ggggatgact atactagccg cagaaaaatc agaacgctga cagatatgactcaggatgaa 2340 attagacaaa gatgggaaag gggatactgt gaccctttca ttgactcaggaagtgactca 2400 gatggacccc tgtaaaagcc acaagcagta aaagtgtgtt aacactttagacagtattat 2460 atttgcttaa gcattaaaag ctttcatata ctcagtagct gtttcacaatcaacaaaaca 2520 atgatgatgt aatcataagg aagtagttta aaataggtta agtaagtttactgcagtaga 2580 taatccctgg ggaggatctg gctctgtaag ctggaacagc aatgttttcagttccagtcc 2640 tctcaaagga gaaccacagg gatgatgcgt tagttcgaat cccattatcctcatggttcc 2700 cttttccaca ttttataatg taagttttag ggataagttt tatatgagctttactaatcc 2760 ttgaaggaag aatagctctt caggtaaaga ggccagtatt aaaagagctacccttccttt 2820 ttatataagg atcgaaacct gctttacgtt ttgctttaat gaagctaagttttaagtttt 2880 aacaggaaat gctcttaggc agaaggtagc tccctcgact gggtgacaagaagaaaccat 2940 tcaggaagtg cttccttctg ctcggggaga tacatgagta tacagtagttcgagtcctgt 3000 gtgctgatgt tgtcttctcg gctcttttgt gacatcacaa atgtaacctagaggtagtct 3060 tttaagaggt ttttacctca ggaaggagtg tggctaatac tggtaaggcacctaactatc 3120 agtacattta agtccattcc tccccatgtt ctcagggtgt gtgcggcgtaagatcgaatc 3180 cccacacacc cgggaacttg cttattgcat aacgttttta ttagtcatatagaaaataat 3240 ataggataag agataggaat taaagcatga ggtgtgtggc tcaacacgtagagtgacaag 3300 gaactctact gtaataggac acaacacctc taaagttgcc cgtgggaaggtgaagtgaga 3360 tcgaatcttt ccttaacgca ggcagctttt tatccactag ggataatgttttaaggaata 3420 ttatagtaat agattgatag ctttaacaat gttagaaata gtatataggaataagatgta 3480 gattgtacga gagctcctca ctactcgctg cgtcgagagt gtatgagactctccaggttt 3540 ggtaagaata tttttattgt tattatgatc cattaa 3576

What is claimed is:
 1. A spumavirus isolated from a human, comprisingSEQ ID NO:
 1. 2. The spumavirus of claim 1, having ATCC Deposit No. ATCCVR-2635.
 3. The spumavirus of claim 1, further comprising a nucleic acidselected from the group consisting of SEQ ID NO. 3, 5, and
 7. 4. Thespumavirus of claim 3, wherein the spumavirus comprises SEQ ID NOs. 1and
 3. 5. The spumavirus of claim 3, wherein the spumavirus comprisesSEQ ID NOs. 1 and
 5. 6. The spumavirus of claim 3, wherein thespumavirus comprises SEQ ID NOs. 1 and
 7. 7. The spumavirus of claim 3,wherein the spumavirus comprises SEQ ID NOs. 1, 3, and
 5. 8. Thespumavirus of claim 3, wherein the spumavirus comprises SEQ ID NOs. 1,3, and 7.